Antibodies which bind an isolated 55 to 75 KDA protein which binds to prion protein

ABSTRACT

The invention involves isolated anti prion protein binding proteins which have molecular weights of from about 55 kD to about 72 kD as determined by SDS-PAGE. Also described is a peptide derived from an isolated anti protein binding protein. Diagnostic uses for each of these molecules are discussed.

RELATED APPLICATIONS

This application is a 371 of PCT/US96/05028, filed Apr. 11, 1996, whichis a continuation-in-part of application Ser. No. 08/421,059, filed Apr.12, 1995, now U.S. Pat. No. 5,679,530, and incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the identification and isolation of proteinswhich bind with prion protein, referred to hereafter as PrP. Moreparticularly, the isolated proteins of the invention has a molecularweight of from about 55 kD to about 72 kD as determined by SDS-PAGE, andare referred to hereafter as anti-PrP proteins or PrP binding protein.Also described is an isolated peptide consisting of an amino acidsequence from said binding protein/anti-PrP protein. Both the peptideand the protein have various diagnostic efficacies. In the case of thepeptide, it can be used, e.g., to produce antibodies which are in turnused to identify the anti-PrP protein. Also, the peptide can bind,itself, to PrP. Similarly, the full protein may be used in the same way.Various diseases associated with prions can thus be diagnosed orscreened using these materials. Further, one can screen for the presenceof PrP in a sample using the protein of the invention.

BACKGROUND AND PRIOR ART

"Prions" or "protein infectious particles", have been implicated in anumber of pathological conditions. Known prion associated diseases arereferred to generally as spongiform encephalopathies, due to a commonfeature of the diseases, i.e., the formation of "holes" in cranialtissue.

By far the most commonly recognized disease associated with prions is"scrapie", found in sheeps and goats. Afflicted animals losecoordination, and eventually become unable to stand. Additional animaldisorders associated with prions include transmissible minkencephalopathy; chronic wasting disease of mule, deer and elk; felinespongiform encephalopathy; and bovine spongiform encephalopathy ("madcow disease"). Among humans, Kuru, or "laughing death" has beenassociated with cannibalism. By far the most serious human disorderassociated with prions, however, is Creutzfeldt-Jakob disease. Thiscondition generally becomes evident via the development of dementia inthe subject. It is a cause of great concern because it can betransmitted iatrogenically, such as by corneal transplantation, use ofcontaminated surgical instruments, injection of purified growth hormonesor other pituitary based materials, as well as via implantation of duramater or electrodes in the brain. Additional pathological conditionsassociated with prions include Gerstmann-Straussler-Scheinker disease(lataxia and cerebellum damage), and fatal familial insomnia. Both ofthese conditions are inheritable, and typically appear in midlife.

At first, the aforementioned conditions were believed to be caused by aslow acting virus found in cerebral tissue. This hypothesis was basedupon the observation that the diseases could be transmitted by injectionof brain extracts of afflicted animals into healthy animals. Thishypothesis, however, is generally no longer accepted, because a virushas not been isolated in spite of concerted efforts to do so.

What has been found about these conditions is that, althoughinheritable, they are caused by proteinaceous material, rather than bynucleic acids. The proteinaceous material is referred to as the prion.Among the experiments which led to the hypothesis that protein materialwas implicated was the treatment of materials from infected animals toinactivate proteins but not nucleic acids. Under these conditions, thedisease was not transmitted.

Elaborations on this hypothesis have identified a single protein inscrapie prions. This protein, "prion protein", will be referred toas--PrP--hereafter. It is used, generically, to refer to the proteinwhich forms the prion. See, e.g., Prusener, Science 252: 1512-1522 (Jun.14, 1991) ("Molecular Biology of Prion Diseases"); Prusiner, et al, ed.,Prion Diseases of Humans And Animals (Ellis Horwood, 1992).

As with all proteins, PrP is encoded by a gene; however, expression ofPrP is not sufficient to cause a prion associated condition. It has beendetermined that PrP may undergo changes in its three dimensionalstructure, leading to its prion form. To elaborate, the benign form ofPrP shows a multiple alpha helix geometry. In the form of infectiveprions, however, the three dimensional structure "elongates", formingbeta sheets. In summary, the difference between the normal, harmlessform of PrP and the form associated with diseases, e.g., appears to becompletely conformational.

"Complementary hydropathy", a concept critical to understanding theinvention described herein, was first suggested by Biro, MedicalHypothesis 7: 981 (1981). The concept Biro set forth was based upon anobservation that protein/protein interactions were observed to bespecific. He argued that complementary coding, i.e., coding by bothsense and "anti-sense" strands of nucleic acid molecules could lead tothe required specificity. Work on the interaction between ACTH,γ-endorphin, angiotensin II, luteinizing hormone release hormone, andfibronectin, and their receptors, supports this hypothesis. See Bost, etal, Mol. Cell Endocrin 44:1 (1986) (ACTH); Carr, et al, J. Neuroimmunol12: 329 (1986) (γ-endorphin), Elton, et al, Proc. Natl. Acad. Sci.U.S.A. 85: 2518 (1988); (angiotensin II); Mulchahey, et al, Proc. Natl.Acad. Sci. U.S.A. 83: 9714 (1986) (luteinizing hormone-releasinghormone); and Brentani, et al, Proc. Natl. Acad. Sci. U.S.A. 85: 364(1988) (fibronectin)

All of this work supported a concept hypothesized by Blalock, et al,Biochem. Biophys. Res. Commun. 121: 203 (1984). Their observation wasthat when the codons for hydropathic amino acids were compared to theircomplementary codons, these complementary codons were generally codonswhich code hydrophilic amino acids. Blalock, et al observed asignificant correlation (r=0.74) between hydropathic coefficients ofamino acids encoded for by opposing DNA strands, and thus postulatedthat peptides encoded by complementary DNA strands would bind oneanother. As indicated, supra, this hypothesis is supported for a numberof peptides.

In 1991, Goldgaber, Nature 351: 106 (May. 9, 1991), reported on thepossible application of complementarily to PrP. Goldgaber reportedanalyzing PrP complementary DNA sequences, and the identification of alarge, overlapping open reading frame in the DNA "antisense" strand forthe PrP gene. When Goldgaber analyzed the deduced amino acid sequencefor this complementary coding region, he found it to be nearly a mirrorimage of PrP. Goldgaber is incorporated by reference in its entirety.While Manson, et al, Nature 352: 291 (Jul. 25, 1991), questioned thiswork, Hewinson, et al, Nature 352: 291 (Jul. 25, 1991) noted that itconfirmed their own work. Prusiner, et al, Nature 362: 213 (Mar. 18,1993), provided an interesting "wrinkle" on this research, when theyreported that they did find an RNA unit of the proper size (4.5 kb) forhybridizing to PrP sense strands, but it was not derived from theantisense PrP strand.

The reports discussed supra, as well as a report by Moser, et al, Nature362: 213 (Mar. 18, 1993), discuss the possibility of the anti-PrPprotein, as it will be referred to hereafter, in prion associateddiseases. Hewinson, et al suggested that the complementary protein mightbe a PrP receptor.

The work which follows presents, for the first time, the identificationand characterization of an anti-Prp binding protein. This material maybe used to identify the presence of PrP in samples, thus providing amethod of screening and/or diagnosis, especially when other symptomscharacteristic of a prion associated disorder are observed. In view ofthe prelevance of prion associated disorders in livestock, e.g., thereare both human and veterinary uses for the invention.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the results of an immunofluorescence assay, usingantibodies produced with the peptide of the invention.

FIG. 2 depicts analyses on SDS-PAGE. In lane A, eluates from cells whichwere negative in the immunofluorescence assay are presented. Lane B wasobtained using eluates from positive cells. Lane C is a Western blot ofan eluate of lane B, using normal mouse serum, while lane D resultedfrom testing such an eluate with the antiserum obtained using thepeptide of the invention.

FIGS. 3A and 3B present data on immunofluorescence assays on murinemesencephalic neurons. In FIG. 3A, the binding of labelled antibodies tocells is shown. FIG. 3B shows localization of the binding.

FIGS. 4A and 4B set forth data regarding immunoelectro-photochemicalcharacterization of prion binding proteins.

FIG. 5 shows the results of experiments establishing prion binding tocomplementary protein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

In order to approach the question of whether an anti-prion proteinexists, a peptide was synthesized, based upon the work of Forloni, etal, Nature 362: 543-546 (1993), the disclosure of which is incorporatedby reference in its entirety. Forloni et al disclose prion peptide "PrP106-126", which induced cell death in primary rat hippocampal cultures.The peptide, which was used herein, i.e.:

Tyr His Val Ala Thr Lys Ala Pro His His Gly Pro Cys Arg Ser Ser Ala (SEQID NO: 1)

is complementary to a peptide derived from PrP, and is itself derivedfrom complementary amino acids 113-129 of a deduced anti-prion ORF,supra.

The synthesized peptide was coupled to Keyhole limpet hemocyanin (KLH),to produce an immunogen. The immunogen was then injected into mice,intraperitoneally, at two week intervals, to a total of 100 ug of totalprotein. After the fourth injection, animals were bled, and titeredagainst uncoupled peptide, via a standard ELISA.

Example 2

Once the antiserum was made, it was utilized in immunofluorescencestudies. Murine neuroblastoma cell line "neuro 2A" was used; Samples ofthe cell line were plated (2×10⁶ cells/well), in eight well tissueculture chamber slides. The cells were incubated, overnight, after whichthe slides were washed and the cells fixed with 1% glutaraldehyde. Afterfixing, a 1:20 horse serum solution was added, for 1 hour at 37° C. Awash with phosphate buffered saline (PBS) followed, and then theantiserum was added, for a period of 2 hours. Following this, the cellswere washed extensively, after which a second antibody, i.e., anti-IgG,coupled to fluorescein isothiocyanate, diluted 1:80 in Evan's blue, wasadded, for 1 hour. Extensive washing followed, after which the slideswere mounted, and observed.

FIG. 1 shows these results, although black and white reproduction doesnot show very clearly the fact that FITC labelling did in fact takeplace (a green color shows that the labelling did take place). Theresults do show that the antiserum recognized the surface of the neuro2A cells. The total reactivity was about 15% of the total population.This result is analogous to results obtained by Butler, et al, J. Virol62: 1558-1564 (1988), who showed that Prp^(sc) infectability in neuro 2Acells was restricted to only certain cells in the population. Theobservation reported herein suggested the next set of experiments,designed to analyze any possible differences between negative andpositive cells.

Example 3

In these experiments, positive and negative cells were first cloned bylimiting dilution. The positive cells derived from clone "IEI2" and thenegative cells from clone "IC4".

Living cells of each clone were surface labelled with ¹²⁵ I, using thewell known lactoperoxidase method. Following labelling, the cells werelysed, and the resulting extracts were incubated with prion peptidePrP¹⁰⁶⁻¹²⁶ coupled to CNBr 4B Sepharose beads overnight, with agitation.

Following the incubation, all bound material (i.e., anything binding tothe PrP¹⁰⁶⁻¹²⁶ peptide), was subjected to SDS-PAGE. To carry this out,the bound materials were separated, using a 40% stacking gel, and a 7.5%resolving gel. Proteins were then transferred to nitrocellulose filters(0.45 um pore size), and were then stained, with 0.5% Ponceau S, toverify extent of transfer.

FIG. 2 presents these results. Lane A is from the negative clone, andlane B from the positive clone. The eluates, when compared, revealedthat a band for a protein of about 55-65 kD from the positive clones,was not found at a high level with the negative clones. In order toconfirm this result, a Western blot was then carried out, as describedbelow.

Example 4

Cells from the positive clone were treated, as in example 1, but werenot labelled. Also as in Example 3, the unlabelled protein extract wasincubated with the PrP¹⁰⁶⁻¹²⁶ Sepharose beads, and bound protein wasthen eluted and applied to filters, also as described. For the Westernblots, the filters were blocked with 5% fat dry milk in PBS, and thenincubated with either normal mouse serum, or the antiserum describedsupra, and then goat anti-mouse biotin conjugated antibody. Thislabelled antibody was added for 1 hour at room temperature. Afterextensive washing, the antibodies were developed, using a well known ECLchemiluminescent system. The results are depicted in lanes C-D of FIG.2, with lane C obtained using normal serum, and lane D the antiserumagainst anti-prion peptide described supra. The findings suggest thatthe antiserum against anti-prion peptide recognizes the PrP¹⁰⁶⁻¹²⁶binding band.

Example 5

The examples set forth supra include analysis of co-cultures of neuronsand glial cells. The possibility of glial cell labelling could not bediscounted, and thus, a protocol was developed to test this possibility.

Glial cells were grown, in culture, using standard methodologies. Thecultured cells were then lysed, and extracts were used in Western blotanalyses, using the antiserum described supra. The glial cells werecompletely negative, as compared to the results which are depicted inFIG. 1. One may infer from this that the target of the antiserum is amolecule on neurons, i.e., it is a nerve cell antigen.

Example 6

Neuronal cells from the mesencephalon of 14 day old Swiss mice embryoswere prepared, in accordance with Mouro Neto, et al, EMBO J 2: 1243-1248(1983), incorporated by reference. These cells were placed onto 10 mmdiameter glass coverslips which had been coated previously withpolyornithine (MW:41 kD; 1.5 ug/ul), in a mixture of DMEM and F12 media,augmented with glucose, glutamine, sodium bicarbonate, and 10% fetalcalf serum. See Garcia-Abreu, Neuros. Res. 40: 471-477 (1995), in thisregard. After 24 hours, extensive neuromal development could be seen.

Murine antiserum, raised against an amino acid sequence consisting ofall but the N-terminal Tyr of SEQ ID NO: 1, and prepared as described,supra, were added at 1:100 v/v, so as to bind to living cells, inaccordance with Halftler et al., Eur. J. Neurosci 1: 297-308 (1989).After one hour of incubation, any excess antiserum was removed, and thecells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH7.6 for 30 minutes. Cells were washed rapidly with 0.1% Triton X-100 inPBS, and incubated overnight with a monoclonal antibody againstβ-tubulin III, diluted 1:100. Incubation with antibodies labelled withFITC and RITC for two hours followed. Cover slips were then mounted inglycerol-propylgallate/PBS (9:1, v/v). The slides were then observed ona Zeiss, axioplan microscope, with an epi-fluorescence attachment.

FIG. 3A shows that the antibodies reacted with all murine neuron cells,which is consistent with recognition of a prion receptor/bindingprotein, expressed by neuron cells. The identify of the cells as neuronswas verified by staining with the mAbs to β-tubulin III.

The coverslips were then studied using a Zeiss Laser Microscope, forwhich a confocal section is shown in FIG. 3B. This localized the boundmolecule to the neuronal membrane.

Example 7

Additional studies were then carried out to identify the bindingmolecule, and to confirm its cellular location.

A total mouse brain extract was prepared, by homogenizing murine brainsin 50 mM Tris•HCl, pH 7.4, 0.2% sodium deoxycholate, 0.5% Triton X-100,1 mM aprotinin, 1 mM leupeptin, 1 mM PMSF, and 1 mM benzamidine, whichwas then centrifuged at 12,000×g for 30 minutes.

A membrane extract was prepared by homogenizing brains in 10 mM Hepes,pH 7.4, 0.5 mM MgCl₂, 1 mM aprotinin, 1 mM leupeptin, 1 mM PMSF, and 1mM benzamidine, followed by centrifugation at 600×g, 15 minutes.Supernatant was then diluted, five times, with the same buffer plus 0.7mM EDTA, followed by centrifugation over a 0.3 M sucrose cushion at825×g for 15 minutes. The pellet was resuspended in 1.38 M sucrose, andcentrifuged under a 0.3 M sucrose solution at 105,000 xg for one hour.The interface was collected and suspended in 20 mM Tris•HCl, pH 7.4, 120mM NaCl, 1 mM leupeptin, 1 mM PMSF, 1 mM aprotinin, and 1 mMbenzamidine.

A cytoplasmic fraction was prepared by homogenizing brain in 50 mMTris•HCl, pH 7.4, 1.5 mM MgCl₂ , 10 mM KCl, 1 mM leupeptin, 1 mM pMSF, 1mM aprotinin, and 1 mM benzamidine, and centrifugation for one hour at105,000×g. The supernatant served as the cytoplasmic fraction.

Samples from each treatment protocol were resolved by SDS-PAGE, andtransferred to a nitrocellulose membrane. Blots were exposed to serumproduced as described supra (odd numbered lanes in the figures, asdiscussed infra), or mouse non-immune serum (even numbered lines, asdiscussed infra).

In FIG. 4A, lanes 1 and 2 show Western blots of total extract; lanes 3and 4 are Westerns for membrane extracts, and lanes 5 and 6 refer tocytoplasmic extracts. As will be seen, a signal at 60 kDa was found intotal brain extracts, and a signal was also found in membrane extracts,but at 63-66 kDa. There was also a detectable species in the cytoplasmicfraction at 60 kDa. See lanes 1, 3 and 5. This suggests that two formsof protein are present in the brain cells, with the membrane bound formbeing somewhat less prevalent, and thus only detectable in purifiedfractions. No signal was found when non-immune serum was used.

In FIG. 4B, analysis via Western blotting after two dimensionalelectrophoresis is shown. Extracts were prepared, as described, supra,and were then isolated, in accordance with O'Farrell et al., Cell 12:1133-1142 (1977), using an ampholyte gradient with light parts at pH5.0-8.0, and two parts of a ampholite gradient at pH 3.0-10.0 in thefirst dimension, followed by 10% SDS PAGE under reducing conditions inthe second dimension and then transfer to nitrocellulose membranes.Total brain extract is in panel 1, and membrane extract in panel 2.Again, serum prepared as discussed supra was used, followed byanti-mouse IgG, labelled with alkaline phosphatase. Substrate for theenzyme was 5-bromo-4-chloro-3-indolyl phosphate, and nitrobluetetrazolium chloride.

FIG. 4B confirms the molecular weights of 60 and 63-66 kDa, with pIs of6.9 and 6.8 in total extract. The membrane fraction showed a singlecomponent (63-66 kDa, pI 6.8).

Example 8

A crucial experiment was to determine if prions bind to the moleculesidentified supra. To determine this, the total brain extract discussedsupra was precipitated, successively, with 30%, 45% and 55% ammoniumsulphate. Pellets were dissolved, subjected to SDS-PAGE and thentransferred to nitrocellulose membranes. Western blots were then carriedout using anti-prion rabbit serum, or serum against the peptidediscussed supra. The membranes were developed using enhancedchemiluminescence, and exposed to X-ray film. (The odd numbered lanes ofFIG. 5 show the anti-prion rabbit serum work, while the even numberedlanes show the anti-complementary peptide antiserum work). It will beseen that the prion proteins were in the 30% fraction (lane 5), andcomplementary proteins in the 55% fraction (lane 2). Lane 2 also showsthat the fraction precipitated between 45 and 55% ammonium sulphatesaturation contained both forms of protein which react with theantiserum against complementary prion peptide. No prion protein wasfound in the 45-55% fraction, as per lane 1. The fraction shown in lane5 did not contain material reactive with anti-complementary peptideantiserum. When the 30% fraction was incubated with electrophoreticallyseparated proteins from the 55% fraction, there was detectablereactivity with rabbit anti-prion antiserum, at a 63-66 kDa molecularweight. See lane 3. This indicates that the normal prion protein, i.e.,PrPc binds to the heavier form of the molecule, i.e., that found in themembrane. Conversely, when the 55% fraction was incubated withelectrophoretic-ally separated proteins of the 30% fraction (lane 6),anti-complementary peptide antibody binding was found, with the bindingbeing to the three known prion isoforms, at 30-33 kDa. Thus, thereceptor protein was able to bind to prions in this assays. These assaysare referred to as overlay assay. Specifically, in the first one, thesolubilized 30% fraction (20 mM Tris•HCl pH 7.4, 120 mM NaCl, 0.05%Tween), was incubated with blots of the 55% fraction on SDS-PAGE. Thesame solution, but using the 55% fraction, was then applied to the 30%blotted fraction.

The foregoing examples set forth a peptide complementary to a peptidefound in PrP. The PrP peptide is known, and is known to be neurotoxic.The inventive peptide, set forth in SEQ ID NO: 1, has been used todevelop antibodies which can be used to identify neurons, since thetarget of the antiserum is specifically nerve cells.

Also a part of the invention are isolated, anti-PrP proteins, alsoreferred to as isolated, PrP binding proteins, which may comprise SEQ IDNO: 1 as part of its amino acid sequence, and which has a molecularweight of from about 55 kilodaltons to about 72 kilodaltons asdetermined by SDS-PAGE. The proteins, given their ability to bind PrP,are useful diagnostically.

As noted, the isolated 55-72 kD protein may be used to determine PrP ina sample. The methodology involves contacting a sample with a 55-72 kDprotein to form complexes therebetween, followed by detection of thethus formed complex. The 55-72 kD proteins may be immobilized, on abead, column glass tube wall, and so forth, but need not be. If notimmobilized, when complexes form in solution, these can be determined byobserving migration patterns on a gel, or by way of any of the standardmethodologies known to the art. Also, any isolated proteins may belabelled, such as with a chromophore, a radiolabel such as ¹²⁵ I, anenzyme, or any of the other standard labels used for determiningbinding. Presence of PrP in a sample may indicate the presence orpredisposition toward a prion associated disorder, such as thosedescribed supra. These diagnostic assays and systems may be used in thecontext of animal husbandry, veterinary medicine, and, of course, humanpathologies and/or general diagnostic assays.

As noted, the peptide of the invention may be used per se in diagnosticmethods, or as an immunogen. In the latter case, it may be coupled to acarrier, such as keyhole limpet hemocyanin, bovine serum albumin, or anyof the standard materials used to "haptenize" small peptides. Theresulting complexes comprising SEQ ID NO: 1, or the peptide per se, maybe formulated in immunogenic compositions, such as with an adjuvant. Asnoted, the antibodies be they polyclonal in the form of antiserum, e.g.,or monoclonal, prepared using standard techniques, which are producedfollowing immunization with the peptides, can be used to detect nervecells carrying the anti-PrP protein.

It has also been found that fusion protein of a first protein and prionprotein can be made, such as "GST-prion". These fusion proteins are alsouseful in carrying out assays for anti-prion peptides and proteins,receptors, and so forth.

Other aspects of the invention will be clear to the artisan, and neednot be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES:  1                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:                           - Tyr His Val Ala Thr Lys Ala Pro His His Gl - #y Pro Cys Arg Ser Ser         #                15                                                           - Ala                                                                         __________________________________________________________________________

We claim:
 1. Antiserum useful in identifying nerve cells which presentan anti-prion protein (anti-PrP protein) on their surface produced byimmunizing a host animal with an isolated peptide comprising the aminoacid sequence set forth in SEQ ID NO: 1 and isolating antiserumtherefrom.
 2. Antiserum useful in identifying nerve cells which presentan anti-PrP protein on their surface produced by immunizing a hostanimal with an isolated peptide comprising the amino acid sequence setforth in SEQ ID NO: 1 complexed to a carrier and isolating antiserumtherefrom.
 3. An isolated monoclonal antibody which specifically bindsto an isolated peptide comprising the amino acid sequence set forth inSEQ ID NO: 1.